N-methyl-D-aspartate (NMDA) receptors are postsynaptic ligand-gated ion channels that mediate excitatory synaptic transmission in the CNS. NMDA receptors are heteromultimers comprised of 2 glycine-binding NR1 subunits and 2 glutamate-binding NR2 subunits, of which there are four types (NR2A,B,C,D). NMDA receptors are involved in normal brain functions such as development, learning, and memory. In addition, NMDA receptor hypo-function may contribute to neuropsychiatric disorders such as schizophrenia, which has lead to the hypothesis that potentiation of NMDA receptor function could be therapeutically useful. However, no small molecule NMDA receptor potentiators exist with which to obtain proof-of-concept data. Given the lack of subunit- selective tool compounds, our objective in this proposal (in response to PAR-09-251 Optimization of small molecule probes for the nervous system, a reissue of RFA-NS-09-003) is to identify potent, subunit-selective potentiators of NMDA receptors that can be used to test specific hypotheses about NMDA receptor function in neurological diseases. To accomplish this, we developed an assay for non-competitive allosteric modulators of NMDA receptors and screened ~100,000 compounds. We identified 6 distinct but related molecules that act at a similar site to potentiate NR2C/D-containing NMDA receptor function; one scaffold appears to favor NR2C potentiation. Our working hypothesis is that these multiple scaffolds can serve as a starting point for the development of potent subunit- selective NMDA receptor potentiators. We propose to use medicinal chemistry to develop a compound with an EC50 value below 300 nM, maximal potentiation of > 2.5-fold, selectivity against other glutamate receptors > 200-fold, solubility > 10-fold EC50, lack of neurotoxicity and selectivity against 72 other CNS receptors, channels, and pumps of between 30-1000 fold. Experiments will address two questions: 1. What structural features control potency and efficacy of NR2C/D-selective NMDA receptor potentiators ? We will use parallel medicinal chemistry approaches to synthesize and test 200 analogues per year, a number supported by our preliminary data. The EC50 value for each compound will be determined at recombinant NR1/NR2A, NR1/NR2B, NR1/NR2C, NR1/NR2D, GluR1 receptors using 14 automated two-electrode voltage-clamp recording systems that can record/analyze concentration-effect curves for > 500 compounds/yr. Preliminary studies have identified several regions of the scaffold that control potency and subunit-selectivity with available chemical space. 2. What are the off-target liabilities and pharmacokinetic properties of NR2C/D potentiators? We will determine solubility and metabolic stability for all active compounds, and screen the best compounds at key decision points and again at the completion of the study against 72 receptors, channels, and transporters using a combination of automated multi-well assays both in the lab and at an NIMH-funded off-target screening center. This information will help to identify the best scaffold to pursue (year-1) as well as characterize the two best compounds to emerge from this study (year-2). We will evaluate plasma half-life, brain:plasma ratio, and neurotoxicity in vitro.